Cyclic nucleotide-modulated channels play major roles in pacemaking activity in heart and brain as well as in olfactory and visual signal transduction in the nervous system. Defects in the functioning of these channels lead to diseases such as epilepsy, cardiac arrhythmia, and color blindness. The overall objective of this grant is to understand how binding of cyclic nucleotides gates (opens/closes) the channels at the molecular level and how lipids modulate the open-closed equilibrium. We will accomplish this by combining state-of-the-art techniques like single-particle cryo electron microscopy (croEM) with high-speed atomic force microscopy (HS-AFM) and functional assays like single-channel electrophysiology and stopped flow fluorescence assays. Our first aim is to determine using single-particle cryoEM high-resolution structures of these channels, in different conformations (no ligand bound, cAMP-bound, cGMP-bound) and with different lipids (by varying the lipid composition in nanodiscs) and assigning these structures to specific ion channel functional states determined using single-channel electrophysiology. Using lipid bilayer single-channel recordings of the channels in various lipid environments, we will assign functional states (open, closed, etc) to the structures and conformations obtained. This aim will not only yield the first ever atomic-resolution structure of a cyclic nucleotide- modulated channel but also structures of other conformations that will allow us to initiate the building of a structural gating model. Our second aim is to determine the various channel conformations in close-to-native conditions (channels reconstituted in lipid bilayers and in physiological buffer at ambient temperature and pressure) using AFM imaging (including HS-AFM). We will determine the conformational landscape at steady states (i.e. in the presence or absence of ligand) as well as the conformational changes that these channels undergo in real time upon ligand binding and how the equilibrium changes with different lipids. We will directly compare these conformations with those obtained by cryoEM in Aim 1. Using stopped-flow fluorescence macroscopic assays of the channels in liposomes, we will investigate how the activation/inactivation kinetics compares with the real time conformational dynamics measured by HS-AFM. The final goal is to formulate structural gating models for cyclic nucleotide-modulated channels using the conformations determined in aims 1 and 2 with assigned functional states.